WO2002033041A2 - Production de production de lysozyme bovine au moyen de vecteurs viraux vegetaux - Google Patents

Production de production de lysozyme bovine au moyen de vecteurs viraux vegetaux Download PDF

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Publication number
WO2002033041A2
WO2002033041A2 PCT/US2001/032147 US0132147W WO0233041A2 WO 2002033041 A2 WO2002033041 A2 WO 2002033041A2 US 0132147 W US0132147 W US 0132147W WO 0233041 A2 WO0233041 A2 WO 0233041A2
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WO
WIPO (PCT)
Prior art keywords
lysozyme
plant
protein
bolys
bovine
Prior art date
Application number
PCT/US2001/032147
Other languages
English (en)
Other versions
WO2002033041A3 (fr
Inventor
Gregory Pogue
Sharlene Velichko
Original Assignee
Large Scale Biology Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Large Scale Biology Corporation filed Critical Large Scale Biology Corporation
Priority to AU2002211748A priority Critical patent/AU2002211748A1/en
Publication of WO2002033041A2 publication Critical patent/WO2002033041A2/fr
Publication of WO2002033041A3 publication Critical patent/WO2002033041A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance

Definitions

  • the present invention is directed to viral vectors and methods for producing transgenic plants that express heterologous DNA that encode a ruminant lysozyme, preferably bovine.
  • This lysozyme protects against diseases caused by plant pathogens, particularly bacterial pathogens.
  • This invention is also directed to methods for producing a ruminant lysozyme from transgenic plants that express the lysozyme.
  • PD spread by sharpshooter leafhoppers, threatens the vine industiy of California.
  • PD is only known from North America through Central America and has been reported in parts of northwestern South America.
  • Xylellafastidiosa is a highly specialized pathogen responsible for this disease. It multiplies in the foregut of sharpshooter leafhoppers, which feed on sap in a plant's xylem, the main water-conducting tissue.
  • the insect carrier delivers bacteria directly into the xylem system of host plants. There they multiply, forming a polysaccharide matrix within the xylem, which eventually blocks the flow of nutrients and results in tissue death.
  • Symptoms include chlorosis and premature production of fruits which are small, tough and, therefore, worthless. The disease is potentially devastating; the most effective control is to produce healthy bacteria-free material for plant propagation.
  • other strains of the pathogen cause far ranging disease in citrus, nut, fruit and other ornamental plants cultivated in the Western Hemi
  • the sharpshooter also exhibits winter-feeding that will encourage vine-to-vine transmission.
  • One additional trait of this sharpshooter that makes it problematic is its promiscuous feeding habits. It feeds on many herbaceous and tree species propagated for ornamental and orchard purposes. This leads to an extensive set of hosts that can be carriers for the Xylella pathogen. Feeding of populations of sharpshooters results in transmission and infection between many species that are growing in the vicinity of an infected plant. Indeed, many worst-case predictions have proved true.
  • the glassy winged sharpshooter has rapidly migrated from the Temecula Valley to virtually every county in California that propagates vines. The presence of the vector and the ubiquitous presence of potential carrier hosts for the bacterium present a large challenge to the entire California wine industry as well as growers of citrus and other valuable host plants.
  • Ruminant gut lysozymes A ruminant is a cud-chewing mammal with two stomachs: a foregut in which anaerobic gram-positive bacteria digest cellulose, thereby permitting the ruminant to use cellulose as a source of energy and nutrients; and a true stomach. Ruminants, such as domestic cattle and other cud-chewing mammals in the order Artiodactyla have developed a symbiotic relationship with bacteria that live in the rumen thereby permitting ruminants to use cellulose as a major nutrient. The bacteria digest cellulose and other dietary components and rapidly grow and divide to large numbers. They convert a significant percentage of the nutrients that are ingested by the ruminant.
  • oligonucleotides were designed that allowed amplification (by polymerase chain reaction - PCR) of the gene from the donor plasmid with restriction sites at either end of the PCR product.
  • the gene fragment was digested with appropriate enzymes (Pac I and Xho I) and ligated into the LSBC GENEWARE® vector pBTI 735 that was similarly digested to yield pi 044 BoLys.
  • GENEWARE refers to a family of expression vectors based on virus genome components and that are capable of autonomous amplification.
  • the resulting ligation was transformed into DH5 alpha E. coli cells and resulting colonies were picked, grown and the DNA purified.
  • the lysozyme substrate used in this assay is 4-methylumbelliferyl-B-D-N, N'-N" triacetylchitotriose.
  • the lysozyme cleaves the substrate, releasing the methylumbelliferone, a substance that is excited at 360 nm and fluoresces at 460 nm.
  • the lysozyme sample is incubated with the substrate in a pH 5 buffer for 1 hour at 42°C, and the reaction is stopped using a high pH buffer. Fluorescence is read in a fluorescent plate reader.
  • a three stage process is preferably employed a bench scale 1-5 kg process is performed to optimize purification steps. On the following week, pilot scale optimization will be completed on 150 kg batches; the third week, 09/18/00, 4 days of full-scale "pilot" manufacturing of bolys protein froml50 kg/day are carried out to complete the process. Formulation and testing continues in the following week.
  • Xylella grows very slowly and generally loses pathogenicity if it is passaged more than twice.
  • the initial goal is to establish liquid cultures of Xylella.
  • Turbidimetric assays are designed to test the ability and efficacy of plant derived and control bolys protein to kill Xylella in vitro.
  • bolys protein may be driven by any of a variety of promoters functional in the context of the recombinant plant viral vector and host plant.
  • plant viral subgenomic promoters are used (U.S. Pat. No. 5,316,931).
  • a portion of the plant materials was vacuum infiltrated and centrifuged to remove apoplastic (extra-cellular matrix) fraction of secreted proteins.
  • a second portion of the plant was ground in an aqueous buffer and then centrifuged at -6,000 xg to obtain a profile of the entire set of soluble plant proteins.
  • the protein extracts were then separated on a SDS-PAGE gel to view proteins by molecular weight and stained with Coomassie blue.
  • the gel showing expression pattern of bolys in total soluble proteins is shown in Fig. 3 and those from IF or infiltrated leaf extracts is shown in Fig. 4. Identity of the bolys protein band was confirmed using protein immunoblotting techniques with antibody preparations provided by NewGene.
  • RNA viruses have allowed the life cycle of numerous plant RNA viruses to be extended artificially through a DNA phase that facilitates manipulation of the viral genome. These techniques may be applied by the person of ordinary skill in the art to make and use recombinant plant viruses of the invention.
  • the entire cDNA of the TMV genome was cloned and functionally joined to a bacterial promoter in an E. coli plasmid (Dawson, W.O. et al, Proc. Natl Acad. Sci. USA 53:1832-1836 (1986)).
  • Infectious recombinant plant viral RNA transcripts may also be produced using other well known techniques, for example, commercially available RNA polymerases from T7, T3 or SP6.
  • RNA polymerase and dinucleotide cap m7GpppG.
  • This not only allows manipulation of the viral genome for reverse genetics, but it also allows manipulation of the virus into a vector to express foreign genes.
  • a method of producing plant RNA virus vectors based on manipulating RNA fragments with RNA ligase has proved to be impractical and is not widely used (Pelcher, L.E. et al, EP 67553A2 (1982).
  • Detailed information on how to make and use recombinant RNA plant viruses can be found, among other places, in U.S. Pat. No. 5,316,931 , which is herein incorporated by reference.
  • the macerated material was pressed through a Vincent Horizontal Screw press containing a 0.023 inch perforated screen (Model VP6K28) to remove fibrous material.
  • the resultant "green juice” was adjusted to a pH of 5.0 with H3PO4.
  • the pH adjusted green juice was heated to 47°C by passage through an Alfa-Laval heat exchanger (Model M3VG) and held at this temperature for 5-10 minutes.
  • the green juice was then cooled to 10-15°C by passage through an Alfa-Laval heat exchanger (Model M3VG). Solid ammonium sulfate was added to the heat-treated green juice at a concentration of 15-20%) saturation, dissolved by stirring and incubated for 30 minutes.
  • the bovine lysozyme present in the 100 kDa permeate was concentrated using an Amicon, 40 ft 2 , cellulose acetate, spiral membrane having a 3 kDa molecular weight cut-off. After concentration, ammonium sulfate was removed from the lysozyme present in the 3 kDa retentate by diafiltration with water or buffer.

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

On produit, dans le cadre de cette invention, de la lysozyme bovine dans des végétaux.
PCT/US2001/032147 2000-10-18 2001-10-17 Production de production de lysozyme bovine au moyen de vecteurs viraux vegetaux WO2002033041A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002211748A AU2002211748A1 (en) 2000-10-18 2001-10-17 Production of bovine lysozyme by plant viral vectors

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US24096700P 2000-10-18 2000-10-18
US60/240,967 2000-10-18

Publications (2)

Publication Number Publication Date
WO2002033041A2 true WO2002033041A2 (fr) 2002-04-25
WO2002033041A3 WO2002033041A3 (fr) 2003-07-31

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Country Status (3)

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US (1) US20020104126A1 (fr)
AU (1) AU2002211748A1 (fr)
WO (1) WO2002033041A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015506667A (ja) * 2011-11-25 2015-03-05 ノボザイムス アクティーゼルスカブ リゾチーム活性を有するポリペプチドおよびそれをコードするポリヌクレオチド
CN109355273A (zh) * 2018-11-09 2019-02-19 浙江理工大学 一种来源于三角帆蚌基因的重组溶菌酶
WO2023168234A1 (fr) * 2022-03-01 2023-09-07 Danisco Us Inc. Enzymes et compositions enzymatiques pour le nettoyage

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AR070326A1 (es) * 2008-01-31 2010-03-31 Oregon State Vectores de closterovirus y metodos relacionados
CN105567714A (zh) * 2014-11-07 2016-05-11 西南民族大学 一种重组牦牛溶菌酶及其制备方法和用途
CN114214303B (zh) * 2022-01-26 2023-02-28 西北农林科技大学 一种反刍动物瘤胃原虫特异溶菌酶OCLyz1A及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993005645A1 (fr) * 1991-09-19 1993-04-01 Smart Plants International, Inc. Protection des plantes contre des agents pathogenes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993005645A1 (fr) * 1991-09-19 1993-04-01 Smart Plants International, Inc. Protection des plantes contre des agents pathogenes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WILCOX ET AL.: 'Production and purification of an active bovine lysozyme in tobacco (nicotiana tabacum): Utilization of value-added crop plants traditionally grown under intensive agriculture' JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY vol. 45, no. 7, 1997, pages 2793 - 2797, XP002960956 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015506667A (ja) * 2011-11-25 2015-03-05 ノボザイムス アクティーゼルスカブ リゾチーム活性を有するポリペプチドおよびそれをコードするポリヌクレオチド
US9663775B2 (en) 2011-11-25 2017-05-30 Novozymes A/S Polypeptides having lysozyme activity and polynucleotides encoding same
US9701952B2 (en) 2011-11-25 2017-07-11 Novozymes A/S Polypeptides having lysozyme activity and polynucleotides encoding same
US10039300B2 (en) 2011-11-25 2018-08-07 Novozymes A/S Polypeptides having lysozyme activity and compositions comprising it
US10119130B2 (en) 2011-11-25 2018-11-06 Novozymes A/S Polypeptides having lysozyme activity and compositions comprising it
US10829750B2 (en) 2011-11-25 2020-11-10 Novozymes A/S Polypeptides having lysozyme activity and polynucleotides encoding same
US10829751B2 (en) 2011-11-25 2020-11-10 Novozyems A/S Polypeptides having lysozyme activity and polynucleotides encoding same
CN109355273A (zh) * 2018-11-09 2019-02-19 浙江理工大学 一种来源于三角帆蚌基因的重组溶菌酶
WO2023168234A1 (fr) * 2022-03-01 2023-09-07 Danisco Us Inc. Enzymes et compositions enzymatiques pour le nettoyage

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AU2002211748A1 (en) 2002-04-29
WO2002033041A3 (fr) 2003-07-31
US20020104126A1 (en) 2002-08-01

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